Abstract

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, PFMO, which is identified as an N-oxide derivative, was identical to P3R of liver microsomes. P3R was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3R = PFMO. Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1.

Introduction

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) (Fig. 1) is an antitumor agent developed in our department (Cholody et al., 1990, 1992). It is a lead compound in the novel class of imidazoacridinones that are inhibitors of both topoisomerases and certain receptor kinases, including FMS-like tyrosine kinase FLT3 (Chau et al., 2006; Skwarska et al., 2010), which exhibited activity against advanced solid tumors under phase I and II of clinical trials and is also being tested for the treatment of autoimmune diseases (Capizzi et al., 2008; Isambert et al., 2010). 5-Diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330) is a less active structural analog of C-1311 (Cholody et al., 1996).

Unlike other antitumor agents, C-1311 expresses only limited mutagenic potential (Berger et al., 1996) and has a low potency to generate oxygen-free radicals, which suggests a lack of cardiotoxic properties. Cellular uptake of this agent occurs rapidly (Burger et al., 1996), and most of the drug accumulates in the nucleus (Skladanowski et al., 1996), which is believed to enable its myeloperoxidase-mediated metabolism and rapid interaction with DNA (Mazerska et al., 2001).

Previous studies on the biological action of C-1311 showed that at low doses this drug induced arrest of the cell cycle progression in the G2 phase and subsequent apoptosis of murine leukemia L1210 cells (Augustin et al., 1996; Lamb and Wheatley, 1996). In ovarian and osteogenic sarcoma cells, G2 arrest resulted only at a low level of apoptosis (Zaffaroni et al., 2001), whereas in the human colon carcinoma HT-29 cell line, drug-treated cells progressed into mitosis after an initial G2 arrest but were unable to undergo cytokinesis and died in a process resembling mitotic catastrophe (Hyzy et al., 2005). Likewise, the treatment of human leukemia MOLT4 cells with C-1311 resulted in mitotic catastrophe, leading to a massive apoptotic response (Skwarska et al., 2007).

Studies on the molecular mechanism of the antitumor action of C-1311 showed that C-1311 intercalated into DNA (Dziegielewski et al., 2002). Furthermore, under oxidative enzymatic conditions, intercalation of C-1311 into DNA is followed by peroxidase-mediated activation of the drug, producing intercalated species that might irreversibly bind to DNA (Dziegielewski and Konopa, 1998; Mazerska et al., 2001). Therefore, it was postulated that intercalation in and also covalent binding to DNA, preceded by metabolic activation of C-1311, were significant steps in the biochemical mechanism of its action. Thus, the molecular mechanism of enzymatic oxidative activation of this drug with peroxidases was investigated (Mazerska et al., 2003). Structural studies of metabolites found products of N-dealkylation on the side chain, C0 and C1 and the metabolite of the dimer-like structure, C3, all presented in Scheme 1. The formation of dimer structures seems to be a common characteristic feature of acridinone oxidative transformations (Mazerska et al., 2002). It was suggested that the reactive carbocation formed in the ortho position to the hydroxyl group should be responsible for the formation of such dimer. Therefore, the formation of dimer-like metabolites under in vitro conditions reflects the potent reactivity of this molecule under cellular conditions in vivo. This reactivity should be responsible for the observed high potency of C-1311 to covalent binding with intracellular nucleophiles: proteins and nucleic acids.

The pathway for C-1311 oxidative enzymatic transformations. Nu, nucleophile. [The scheme has been drawn on the basis of Scheme 2 in Mazerska et al. (2003).]

In consideration of the reactivity of imidazoacridinone drugs described above, studies on metabolic transformation of C-1311 and its less active methoxy analog, C-1330, with rat and human liver microsomal enzymes are presented here. We aimed to identify liver enzymes that play a crucial role in C-1311 metabolism in humans. Therefore, HPLC analyses of the incubation mixture obtained with the set of 16 human liver protein probes with known contents of 14 enzymes [reaction phenotyping kit (RPK)], as well as with human recombinant P450 isoenzymes and human recombinant flavin monooxygenases (FMOs) and UDP-glucuronosyltransferases (UGTs), were performed, and structures of metabolites were proposed. To elucidate the role of P450 in liver metabolism, C-1311 was eliminated from mice in which NADPH-P450 oxidoreductase (POR) has been conditionally deleted in the liver. The results obtained will allow the prediction of metabolic pathways of C-1311 in future patients and together with the data on individual isoenzyme levels resulting from the genetic polymorphism and enzyme induction by other xenobiotics will help in the design of individually directed antitumor therapy with this drug.

Reaction Phenotyping Kit.

The RPK (version 6) was purchased from tebu-bio. It contained 16 samples (m) of liver microsomal fractions derived from selected patients. The activities of 10 P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4/5, and 4A11) (xn) were determined by the manufacturer for each sample. The samples were collected for the RPK in such a manner that activities of P450s were nearly without correlation and therefore can be used as independent variables in multiple regression analysis (MRA). The incubation of C-1311 with each of the 16 samples was performed according to the procedure described below for incubation with the liver microsomal fraction. Enzymatic activity of each fraction toward C-1311 was measured as a conversion percentage (ym) determined after 60 min of incubation and expressed as logit(ym) for statistical analysis.

To evaluate the role of a particular P450 in metabolism of C-1311, the correlation studies and MRA were performed. MRA consists of the calculation of regression coefficients a0, a1, …, an, giving the relationship between dependent variable y and independent xn descriptors in the form of an equation: y = a0 + a1x1 + …,+ anxn. The statistical significance of the whole equation was determined by the FSnedecor test. The correct regression equation should contain only terms characterized by significant coefficients. The significance of each coefficient a1, a2, …., an is calculated by Student's t test. Insignificant terms are removed from the equation in a one-by-one manner.

Animals.

All mouse experiments were performed in the Biomedical Research Centre (Dundee, UK), in accordance with the Animal Scientific Procedures Act (1986), after ethical review by the University of Dundee and Cancer Research UK. The generation of hepatic reductase null (HRN) mice has been described previously (Henderson et al., 2003). Male PORlox/lox (wild-type) and PORlox/lox + CREALB (HRN) mice between 12 and 16 weeks of age were used in all experiments. All mice were maintained under standard animal housing conditions with free access to a standard rodent diet and water and a 12-h light/dark cycle.

Instrumentation.

Supernatants obtained according to the procedures described below were analyzed by reverse-phase HPLC instruments equipped with a 5-μm Suplex pKb-100 analytical column (0.46 × 25 cm, C18; Supelco, Bellefonte, PA). Waters (Milford, MA) HPLC systems with a dual λ absorbance detector and Waters 2484 Breeze software or a multidiode array detector and Waters 2996 Millenium software and an Agilent 1100 system (Agilent Technologies, Santa Clara, CA) with ESI-MS detection were applied. Waters 2484 and Millenium 2996 HPLC systems were equipped with a Rheodyne injector 7725i and with a binary pump (Waters 1525) or with a model 600E system controller. The Agilent 1100 HPLC system included a binary pump and autosampler.

Metabolism of C-1311 and C-1330 with Microsomal Enzymes.

Incubations with liver microsomal fractions.

The stock solutions of 2 × 10−3 M C-1311 and C-1330 or 2 × 10−2 M NADPH were prepared in a 0.05 M phosphate buffer, pH 7.4. Incubations of C-1311 and C-1330 with liver enzyme fractions listed above were performed in a 0.05 M phosphate buffer, pH 7.4, with 2 × 10−3 M NADPH, enzyme protein (2 mg/ml) and 2 × 10−4 M C-1311 or C-1330. The incubation was performed at 37°C in air. Preincubation of proteins and the drug for 5 min was followed by the addition of NADPH. After an appropriate period of time, the incubation mixture was placed in ice and an equal volume of ice-cold methanol was added. Then the solution was centrifuged for 15 min at 12,000g. The supernatant was analyzed directly by RP-HPLC. Results representative of at least three independent experiments were considered. The conversion percentages of C-1311 presented in Fig. 3 were calculated as the ratio of substrate HPLC peak area after 0 and 60 min of incubation.

Inactivation of microsomal P450 enzymes with 1-ABT.

C-1311 (2 × 10−4 M) was incubated at 37°C for 60 min with 2 mg/ml microsomal proteins (stock of 20 mg/ml) that were previously preincubated with 10−3 M 1-ABT (stock of 10 mM) at 37°C for 30 min in the presence of 2 × 10−3 M NADPH (stock of 2 × 10−2 M). The final volume of the incubation mixture in 0.1 M potassium phosphate buffer (pH 7.4) was 100 μl. Incubations of C-1311 without 1-ABT served as controls. After an appropriate incubation time, the reactions were stopped by the addition of 100 μl of ice-cold methanol. Then the solutions were briefly centrifuged for 5 min at 12,000g. The supernatants (100 μl) were analyzed by RP-HPLC with UV-Vis detection. All experiments were conducted in duplicate.

Thermal inactivation of microsomal FMO.

Duplicate sets of samples containing 2 mg/ml rat pooled liver microsomes (stock of 20 mg/ml) in a 0.1 M potassium phosphate buffer (pH 7.4) were prepared. One set was supplemented with 2 × 10−3 M NADPH (stock of 2 × 10−2 M), and both sets were incubated at 45°C for 5 min and cooled on ice for 2 min. NADPH was added to the set of samples in which it had been omitted previously. Then 2 × 10−4 M C-1311 (stock of 2 × 10−3 M) was added after preincubation at 37°C for 5 min, and both sets of samples were incubated under regular conditions. The final total volume of the incubation mixture in 0.1 M potassium phosphate buffer (pH 7.4) was 100 μl. Control incubations were not heat-treated.

Incubation with liver microsomal fractions in the presence of NADPH and UDPGA cofactors.

Incubations of C-1311 or C-1330 (2 × 10−4 M) with HLM and RLM (1, 2, or 3 mg/ml) were performed in a 1× UGT buffer mix (50 mM Tris-HCl, 8 mM MgCl2, and 0.025 mg/ml alamethicin in water) at 37°C in a total volume of 50 μl. The incubation mixtures were preincubated for 5 min at 37°C and followed by the addition of UDPGA (1.25, 2.5, or 3.75 × 10−3 M) or UDPGA (2.5 × 10−3 M) and NADPH (10−3 M). Control reactions without microsomes and UDPGA were simultaneously performed. Hydrolysis of glucuronides was performed after a 1-h incubation, and reaction mixtures (50 μl) were supplemented with 10 U/μl of GUS. The reactions were stopped after 1 h by the addition of 50 μl of ice-cold methanol. The solutions were then centrifuged for 15 min at 12,000g. The supernatants (50 μl) were analyzed by RP-HPLC with UV-Vis detection at 380 nm. All assays were conducted in triplicate.

Transformation of Imidazoacridinones with Human Recombinant Enzymes.

Transformations with human recombinant P450s.

Incubations of 2 × 10−4 M C-1311 (stock of 2 × 10−3 M in phosphate buffer) with 200 pmol/ml human recombinant P450s (1A2, 2C9, 2C19, 2D6, and 3A4) and 10−3 M NADPH were performed in a 10−4 M potassium phosphate buffer (pH 7.4) at 37°C in a total volume of 50 μl. The incubation mixtures were preincubated for 5 min at 37°C followed by the addition of NADPH. After the appropriate incubation times, the reactions were stopped by the addition of 50 μl of ice-cold methanol. Then the solutions were briefly centrifuged for 5 min at 12,000g. The supernatants (50 μl) were analyzed directly by RP-HPLC. All assays were conducted in duplicate. To confirm that the incubation system was performing properly, the incubations of all isoenzymes with their standard substrates were also performed according to the procedure described below.

Inhibition of human recombinant P450s activity.

The catalysis of 7-ethoxycoumarin deethylation, β-hydroxylation of testosterone, and transformations of imipramine to N-demethylated analog and 2-hydroxyimipramine by CYP1A2, CYP3A4, CYP2C19, and CYP2D6, respectively, were controlled. Then 2 × 10−5 M testosterone or 7-ethoxycoumarin or imipramine was preincubated for 5 min with 50 pmol/ml selected P450 isoenzyme with or without earlier incubation with C-1311. All reactions were performed in 10−4 M potassium phosphate buffer (pH 7.4) at 37°C in a total volume of 50 μl. The reactions were started by the addition of 1 mM NADPH and stopped by addition of an equal volume of ice-cold methanol. Next, samples (100 μl) were cooled for 10 min and were briefly centrifuged at 12,000g for 5 min. The supernatants were analyzed directly by HPLC/UV-Vis. The rates of substrate metabolism were calculated as a ratio of the surface area under the substrate HPLC peak after and before enzymatic reaction. All experiments were performed in duplicate.

Metabolism by human recombinant FMOs.

Incubations of 10−4 M C-1311 and C-1330 (stock of 10−3 M in phosphate buffer, pH 8.4) with 1 mg/ml human FMO isoenzymes (FMO1, FMO3, and FMO5) were performed in a 10−4 M potassium phosphate buffer (pH 8.4) at 37°C in a total volume of 50 μl. Preincubations of the compound with enzyme were followed by the addition of 3 × 10−4 M NADPH. After an appropriate incubation time, the reactions were stopped by the addition of 50 μl of ice-cold methanol. Then the solutions were briefly centrifuged for 5 min at 12,000g. The supernatants (50 μl) were analyzed directly by RP-HPLC. All experiments were conducted at least in duplicate.

Glucuronidation assay with human recombinant UGT1A1 and UGT2B7.

Incubations of C-1311 or C-1330 (5 × 10−5 M) with the selected human recombinant UGTs, UGT1A1 and UGT2B7 (0.2, 0.5, and 1 mg/ml), were performed in a 1× UGT buffer (50 mM Tris-HCl, 8 mM MgCl2, and 0.025 mg/ml alamethicin in water) at 37°C in a total volume of 50 μl. Preincubations of the compound with enzyme were followed by the addition of UDPGA (2 × 10−3 M). After appropriate incubation time, the reactions were stopped by the addition of 50 μl of ice-cold methanol. The solutions were then centrifuged for 15 min at 12,000g. The supernatants (50 μl) were analyzed by RP-HPLC with UV-Vis detection at 380 nm. All experiments were conducted in triplicate.

HPLC Analyses and Structures of Metabolites.

All reactions were controlled by Waters HPLC systems at a flow rate of 1 ml/min with the following mobile phase system: a linear gradient from 15 to 80% methanol in sodium phosphate buffer (0.05 M, pH 3.5) or ammonium formate (0.05 M, pH 3.4) for 25 min, followed by a linear gradient from 80 to 100% methanol in a sodium phosphate buffer or ammonium formate for 3 min. The studies on the metabolite structures were performed by HPLC-ESI/MS and HPLC-MS/MS analyses with the aid of an Agilent 1100 LS/MSD mass spectrometer.

Pharmacokinetic Studies of C-1311 in HRN and Wild-Type Mice.

Two pharmacokinetic studies using HRN and WT mice were carried out with C-1311. The drug was administered as a single intraperitoneal dose at 50 mg/kg b.wt. (stock of 5 mg/ml in phosphate-buffered saline, using 10 ml/kg). Blood samples (10 μl) were taken from the lateral tail vein at 10, 20, 40, and 60 min and at 2, 4, and 6 h after administration of C-1311 and placed in heparinized vials (containing 10 μl of heparin, 15 IU/ml) on ice. For blood samples, 10 μl of internal standard (warfarin, stock of 10 μg/ml in methanol) was added to each tube. The samples were vortexed for 1 min, followed by 5 min of sonication and centrifuged at 15,000g for 5 min. The supernatants were then transferred directly into HPLC vials and analyzed with ESI-MS detection.

Results

C-1311 and C-1330 Metabolism with Rat Liver Microsomes.

Chromatograms of the incubation mixtures with RLM presented in Fig. 2, A and B, indicate that at least four metabolites were formed after metabolism of C-1311 and C-1330. There is one product of higher concentration, P3R and P*3R, respectively, and three of lower concentrations, P1R, P2R, P4R and P*1R, P*2R, and P*4R for each compound. The labeling of products is presented in Table 1. The comparison of UV-Vis spectra of P1R and P*1R peaks with those of C-1311 and C-1330 substrates presented in Fig. 2C resulted in notable differences. This result indicates that significant changes in the chromophore moiety occurred after metabolism to P1R and P*1R products. ESI-MS spectra recorded for these peaks gave m/z values of 367.1 and m/z 381.1, respectively. They are 16 units higher than those of substrates, indicating that the substitution of a hydroxyl group or addition of an oxygen atom to the parent compound took place. The above spectral data of both metabolites, P1R and P*1R, and the analysis of the chemical properties of C-1311 (Scheme 1), allowed us to propose the substitution of a hydroxyl group in a position ortho to the 8-hydroxy substituent in imidazoacridinone (Fig. 2D).

The ESI-MS spectrum of the P*R metabolite of C-1330 gave an m/z 351.1, 14 units lower compared with that of the substrate. This result indicated, together with the retention time identical to that of C-1311 (Fig. 2, A and B), that P*R was likely to be a demethylation product on the methoxyl group of C-1330 with a structure exactly like that of C-1311 (Fig. 1). The identification of the P3R structure will be described below.

Microsomal proteins from 16 human liver samples (RPK) were incubated with C-1311 under the conditions described under Materials and Methods. Four metabolites were observed during HPLC analyses of all incubation mixtures. Peak retention times and UV-Vis and ESI-MS spectra (data not shown) were identical to those described above for rat microsomes (Fig. 2). Thus, identical metabolites P1 to P4 were formed in rat and human microsomes.

The rates of C-1311 transformations with 16 samples of human microsomal enzymes (RPK) are presented in Fig. 3 as percent conversion of C-1311. The rates of C1311 transformation shown there were widely variable from 3 to 45%. Three samples displayed very low rates and four of them were close to 45%, whereas the remaining samples yielded transformations between 15 and 40%. To elucidate the role of particular P450s in liver microsomal metabolism of C-1311, the values of transformation rates determined above and the activity data of 10 hepatic P450s delivered by the RPK manufacturer were applied for correlation and MRA. The values of correlation coefficients obtained were from −0.274 to 0.193, that is, very low. The “best” regression equation was characterized by the very low value of the FSnedecor test equal to 1.89, and regression coefficients for this equation were insignificant. These results allowed us to postulate that none of the 10 P450s considered play a significant role in metabolism of C-1311 by liver microsomal enzymes.

Transformations of C-1311 with 16 samples of human microsomal enzymes (RPK) after a 60-min incubation. The incubation mixtures contained 0.2 mM C-1311, 2 mM NADPH, and 2 mg/ml proteins in 0.1 M phosphate buffer, pH 7.4. Reactions were terminated with equal amounts of methanol, and after precipitation of microsomal proteins supernatants were analyzed by HPLC via direct injection. The percentage conversion of substrate was calculated as the ratio of substrate HPLC peak area after 0 and 60 min of incubation. The graphs are representative of three independent experiments.

Metabolism of C1311 and C-1330 in the Presence of E. coli Recombinant Human P450 Isoenzymes.

Taking into account that C-1311 and C-1330 were metabolized by human microsomal enzymes and having no evidence that P450 enzymes participated in this metabolism, we tested the susceptibility of these compounds to transformation with individual P450s. Human E. coli recombinant enzymes CYP1A2, 2C9, 2C19, 2D6, and 3A4, the representatives of three P450 families, were incubated with both agents. A unexpected result was that although various reaction conditions were applied and several control experiments were included (see Materials and Methods), no reactivity of imidazoacridinones with P450s was observed and no metabolic products were found (data not shown). Two key reasons for the resistance of C-1311 toward isolated P450s should be considered: either microsomal enzymes other than P450s are responsible for the observed microsomal metabolism or these drugs are able to inhibit P450 enzymes. Both of these possibilities were investigated, and the results are presented below.

Inhibition of P450 Isoenzyme Activity by C-1311 and C-1330.

To evaluate whether C-1311 or C-1330 inhibits cytochrome P450 enzymes, the influence of both agents on metabolism of standard substrates of selected P450s was investigated. The rates of the formation of metabolites (7-hydroxycoumarin, 6β-hydroxytestosterone, and imipramine derivatives) with E. coli recombinant human P450s in the presence and absence of C-1311 were compared and are presented in Fig. 4. It was shown that C-1311 is a strong inhibitor of CYP1A2 isoenzyme in a time- and concentration-dependent manner. It also inhibited the activity of CYP3A4. In contrast, this compound did not inhibit enzymes of the CYP2 family. The comparison of the inhibition ability of C-1311 with those of C-1330 shown in Fig. 5 demonstrated that, like C-1311, C-1330 inhibited the action of CYP1A2 and CYP3A4, albeit to a lesser degree than C-1311. To confirm the above results, metabolic transformations of 7-ethoxycoumarin in the presence of C-1311 were also studied with rat liver microsomes. The results demonstrated that under these conditions C-1311 also inhibited CYP1A2 in similar concentration- and time-dependent manner (data not shown).

Effect of C-1311 on the formation of P450 metabolites from standard substrates: 7-ethoxycoumarin with CYP1A2, testosterone with CYP3A4, and imipramine with CYP2D6 and CYP2C19. Substrates (0.02 mM) were incubated in phosphate buffer, pH 7.4, with 50 pmol/ml E. coli recombinant human P450s of low POR level, with 1 mM NADPH, and without or with various C-1311 concentrations. The amount of the main metabolite as the surface area under metabolite HPLC peak was measured after different incubation times. The results are the medium value of three independent experiments with the S.D. presented.

Comparison between CYP1A2 and CYP3A4 inhibition ability of C-1311 and C-1330. The experimental conditions were identical to those in the legend to Fig. 4. The percentage remaining enzyme activities were calculated as the ratio of the main metabolite HPLC peak surface area after 0 and 30 min of incubation. The graphs are representative of three independent experiments.

Elimination of C1311 in WT and HRN Mice.

In the next step of elucidation of the role of hepatic P450s in metabolism of C-1311, we compared the elimination of the drug with blood and urine occurring in WT mice and in HRN mice, in which POR has been conditionally deleted in the liver (Henderson et al., 2003). It is assumed in experiments of drug elimination in WT and HRN mice that POR associated with cytochrome P450 isoenzymes is necessary for their activity. As a consequence, the finding of better elimination of the drug in WT than in HRN mice indicates that the absence of POR diminished the metabolism of this agent; thus, P450 plays a crucial role in drug transformation, leading to its elimination.

Mice were treated at 50 mg/kg by intraperitoneal injection, and then blood and urine samples were taken at 10, 20, 40, and 60 min and at 2, 4, and 6 h and were analyzed by HPLC. The comparison of elimination between HRN and WT mice is shown in Fig. 6. The results revealed marginally slower excretion of C-1311 in the blood and urine of HRN than of WT mice. The lack of significant differences between HRN and WT mouse elimination strongly indicated that cytochromes P450 have little or no effect on C-1311 pharmacokinetics.

Elimination of C-1311 in blood (A) and urine (B) of WT (□) and HRN (♦) mice. The drug was administered as a single intraperitoneal dose at 50 mg/kg b.wt. Blood samples (10 μl) were taken from the lateral tail after administration of C-1311 and placed in heparinized vials on ice. The samples were vortexed, sonicated, and centrifuged. The supernatants were then transferred directly into HPLC vials and analyzed. The results are the medium value of two independent experiments with the S.D. presented.

Metabolism of C-1311 and C-1330 with FMO.

In the search for microsomal enzymes involved in metabolism of C-1311 other than cytochromes P450, three forms of human recombinant flavin monooxygenases (FMO1, FMO3, and FMO5) were incubated with C-1311 and C-1330. Figure 7 shows that higher transformation rates were observed for both compounds with FMO1 compared with FMO3 after various incubation times. FMO5 was shown to be inactive toward C-1311 and C-1330. Metabolism with FMO1 and FMO3 yielded one main metabolite, PFMO, presented in Fig. 8A.

Metabolism of C-1311 (A) and C-1330 (B) with recombinant human FMO1 and FMO3 isoforms. Each compound (0.1 mM) was incubated in phosphate buffer, pH 8.4, with 1 mg/ml human recombinant FMO1 and FMO3 expressed in baculovirus-insect cells and 0.3 mM NADPH. Percent conversion of the substrate was calculated as the ratio of substrate HPLC peak area after the given and 0 time of incubation. Results are the medium value of three independent experiments with the S.D. presented.

Comparison of HPLC profiles and UV-Vis spectra of metabolites obtained for FMO-mediated C-1311 metabolism with those observed for the mixture of microsomal enzymes (both presented in Fig. 8A) strongly suggested that the identical PFMO and P3R metabolite might be formed in both metabolic systems. ESI-MS analysis in Fig. 9A indicated that PFMO and P3R HPLC bands observed in Fig. 8A gave the identical m/z 367.1 value for molecular ions. This value (M + 1 + 16) is 16 units higher than that of the substrate and suggests the additional oxygen atom in the metabolite. In addition, UV-Vis spectra of PFMO and P3R metabolites shown in Fig. 8B are identical to those of the substrate, which strongly indicates that no changes in the chromophore part of metabolite molecule have occurred. Analogous HPLC, UV-Vis, and ESI-MS analysis was also performed for microsomal and FMO-mediated metabolism of C-1330 (Fig. 9B). Considering the studies presented above and the fact that FMO catalyzes specific oxidation of nitrogen or sulfur atoms to the relative N- or S-oxides, the products P3R/H = PFMO and P*3R/H = P*FMO have been proposed to be N-oxide derivatives on the ω-nitrogen atom of C-1311 and C-1330, respectively. Their structures are presented in Fig. 9, A and B.

Additional experiments were performed to confirm that PFMO = P3R/H is a product of C-1311 metabolism by FMO. HPLC analyses of the metabolite mixtures obtained in all experiments with HLM are summarized in Fig. 10. First, the common inhibitor of cytochrome P450 isoenzymes 1-ABT was added to the incubation mixture of C-1311 with human liver microsomes. As a result, one metabolite identified as PFMO was observed (dotted gray line), whereas the amounts of P1H, P2H, and P4H were negligible. The second experiment was based on the fact that FMO activity is very sensitive to temperatures higher than 37°C, and this phenomenon is observed only in the absence of NADPH cofactor (Cashman, 2008). The presence of NADPH in the reaction mixture before heating protects thermal deactivation of FMO. Figure 10 shows that the heating after preincubation with NADPH (gray line) gave a high concentration of PFMO. However, the temperature increase to 45°C before preincubation with NADPH (dotted black line) evidently resulted in the lack of PFMO, whereas concentrations of the remaining products were very close to that presented before (black line). The results indicated clearly that the enzyme responsible for the formation of PFMO was inactivated under higher temperature. In conclusion, N-oxide derivatives in the side chain of C-1311 and C-1330 were shown to be the metabolites PFMO = P3R/H and P*FMO = P*3R, respectively, formed with FMO as well as with liver microsomal fraction.

HPLC analysis of C-1311 incubated with HLM under standard conditions (black lines) with the inhibitor of P450s, 1-ABT (dotted gray line), with preincubation of microsomal proteins at 45°C before the NADPH addition (dotted black line), and with preincubation of microsomal proteins together with NADPH (gray line). HPLC bands at 16, 22, and 27 min relate to metabolites P1H, P2H, and P4H, respectively. The incubation mixtures contained 0.2 mM C1311, 2 mM NADPH, and 2 mg/ml proteins in 0.1 M phosphate buffer, pH 7.4. Detailed procedures with 1-ABT and under higher temperature were described under Materials and Methods. Reactions were terminated with an equal amount of methanol, and after precipitation of microsomal proteins supernatants were analyzed by HPLC via direct injection. Each chromatogram is representative of three independent experiments.

Glucuronidation of C-1311 with Microsomal Proteins and Recombinant UGT1A1 and UGT2B7.

Incubation of C-1311 with rat and human microsomal enzymes in the presence of UDPGA, the cofactor of UGTs yielded the new product, P5, observed in HPLC chromatograms in Fig. 11A (dotted line). However, C-1330 possessing a methoxyl instead of a hydroxyl group at position 8 did not give such a metabolite (Fig. 11C). The following incubation of the reaction mixture with GUS resulted in the disappearance of P5 (gray line in Fig. 11A). To confirm that glucuronidation of C-1311 takes place with microsomes, the representatives of two UGT families, human recombinant UGT1A1 and UGT2B7, were used for incubation with both compounds. HPLC analysis in Fig. 11B showed that C-1311 was the substrate of UGT1A1 and gave the PUGT metabolite, whereas Fig. 11C showed that C-1330 was resistant to UGT1A. Furthermore, neither compound was sensitive to the action of UGT2B7 (data not shown).

HPLC analysis of C-1311 incubated with RLM under standard conditions (A) (black lines), with the addition of UDPGA cofactor (dotted line), and after treatment with β-glucuronidase (gray line) and human recombinant UGT1A1 and UGT2B7 (B). C, HPLC analysis of C-1330 incubated with RLM under standard conditions (black lines) and with the addition of UDPGA cofactor (dotted line) and with human recombinant UGT1A1 (gray line). The incubation mixtures with microsomes contained 0.2 mM C-1311 or C-1330, 2 mM NADPH, and 2 mg/ml proteins in 0.1 M phosphate buffer, pH 7.4. Detailed procedures with the addition of UDPGA and β-glucuronidase were described under Materials and Methods. Incubations of 0.05 mM C-1311 and C-1330 with human recombinant UGT1A1 and UGT2B7 (0.2, 0.5, and 1 mg/ml) performed in 1× UGT buffer (details under Materials and Methods) at 37°C were followed by the addition of 2 mM UDPGA. Reactions were terminated with an equal amount of methanol, and supernatants were analyzed by HPLC via direct injection. Each chromatogram is representative of three independent experiments.

The comparison of retention times and UV-Vis and ESI-MS spectra of PUGT and P5 observed with recombinant UGT and microsomes, respectively, allowed the conclusion that they were identical metabolites. Their m/z value or 527.2, which was equal to (M + 176 + 1), indicated that PUGT = P5 should be the glucuronide derivative of C-1311. The absence of the C-1330 metabolite under the above conditions is a strong suggestion that glucuronidation of C-1311 occurred at the 8-hydroxyl group. In light of these results, the glucuronidation of both compounds at the nitrogen atoms of the side chains is of rather low probability.

Discussion

After our previous studies on metabolism of C-1311 with peroxidases (Mazerska et al., 2003) and microsomal enzymes (Wiśniewska et al., 2007), the present work was intended to elucidate the role of human liver enzymes in metabolism of this drug.

We showed that C-1311, as well as its less active 8-methoxy analog, C-1330, was metabolized with liver microsomal enzymes, resulting in identical products in rats and humans. P1R and P*1R metabolites (Fig. 2D) were identified as derivatives of C-1311 and C-1330, respectively, with an additional hydroxyl group in the heterocyclic core. The identical P1 product was also found previously in mouse urine and plasma (Calabrese et al., 1999) because hydroxy derivatives are common metabolites formed by aromatic compounds from their epoxide intermediates. The second microsomal metabolite of the methoxy derivative, C-1330, was the demethylation product, P*R, identical to C-1311 (Figs. 1 and 2B). Therefore, C-1330 should be considered as a prodrug of C-1311.

Asking whether any of the cytochrome P450 isoenzyme(s) are preferentially involved in the transformation of imidazoacridinones, we investigated metabolism with 16 human microsomal samples and with human E. coli recombinant CYP1A2, 2C9, 2C19, 2D6, and 3A4. Although various reaction conditions were studied, we showed unexpectedly that cytochrome P450 isoenzymes did not participate in metabolism of this compound. In view of this result, our hypothesis was 2-fold: either C-1311 is able to inhibit P450 enzymes or other microsomal enzymes are responsible for the observed metabolism. Both suggestions were investigated in this work.

The verification of the first hypothesis allowed us to show that CYP1A2 and CYP3A4 were inhibited by C-1311 as well as by C-1330. These results might have a strong impact on the drug-drug interaction in multidrug therapy because C-1311 was applied in phase II clinical trials with anthracyclines (Capizzi et al., 2008) and will be used with other therapeutic agents. Until now the inhibition of selected P450 isoenzymes was reported just for a few antitumor agents, e.g., thiotepa (Jacobson et al., 2002; Richter et al., 2005) and tamoxifen (Sridar et al., 2002; Notley et al., 2005). Other drugs delivered more examples, for instance, carbamazepine (Pearce et al., 2005, 2008) or 17α-ethynylestradiol (Lin et al., 2002). The latter strongly inhibited CYP3A4 and at the same time was being metabolized by it. Thus, imidazoacridinones, particularly C-1311, were shown to be the inhibitor of recombinant human quinone oxidoreductase at a concentration close to that observed here for P450s (Nolan et al., 2010).

In this study, we showed that not only C-1311 but also C-1330 inhibited the action of P450s although it possesses a methyl substituent at the hydroxyl group. On the other hand, we reported earlier that a nonsubstituted hydroxyl group was necessary for peroxidase-mediated metabolism that resulted in covalent binding of the C-1311 metabolite to DNA (Mazerska et al., 2001). Thus, the current results indicated that the mechanism responsible for metabolic activation of C-1311 with peroxidases should differ from that responsible for C-1311-mediated inhibition of P450 isoenzymes. We suggest that the imidazole region of the C-1311 heterocyclic ring would participate in the inhibition mechanism, leading to the formation of a type II complex with the heme of P450. Such a mechanism was shown for the structurally similar pyrimidineimidazole derivative 2-((2-(1H-imidazol-1-yl)-6-methylpyrimidin-4-yl)(3-((benzo-[d][1,3]dioxol-5-ylmethyl) (methyl)amino)propyl)amino)acetamide (PH-302) (Hutzler et al., 2006) and other drugs of heterocyclic structure.

Recombinant human FMO1 and FMO3 but not FMO5 catalyzed the formation of one metabolite, PFMO and P*FMO, for C-1311 and C-1330, respectively, and identical metabolites, P3R and P*3R, were obtained in microsomes (Figs. 8 and 9). Therefore, we postulated that FMOs are responsible for the formation of P3R and P*3R with microsomes. This hypothesis was confirmed by results of C-1311 metabolism with HLM in the presence of P450 and FMO inhibitors (Fig. 10) and in experiments performed with the heating of microsomal enzymes and with the protection of NADPH. In conclusion, we showed that the formation of major metabolites P3R/H and P*3R with rat and human liver enzymes, identified as Nω-oxide derivatives, was catalyzed by FMO.

There are only several reports about the participation of FMO metabolism in detoxification of other antitumor agents such as pyrazoloacridine derivatives (Reid et al., 2004) or tamoxifen (Krueger et al., 2006). It may be advantageous to develop a drug that is metabolized by FMO enzymes but not by P450s. First, it is a way of avoiding P450-mediated bioactivation and a type of adverse drug-drug interactions. Furthermore, the formation of polar readily excreted FMO metabolites has significant clinical utility because they may help to control clearance and lead to more desirable pharmacokinetics. In addition, FMO enzymes are not easy induced or inhibited, because the modulation of FMO is largely due to genetic polymorphism. Therefore, in this case, it is much easier to predict drug metabolism in patients.

Results indicating the lack of P450 reactivity toward C-1311 in vitro were supported by experiments with animals of normal, WT, and diminished (HRN) activity of liver cytochromes P450. The elimination of C-1311 in blood and urine was only slightly slower in HRN than in WT mice. Therefore, the decrease in cytochrome P450 activity did not prevent metabolic transformations of this drug in mice. This result confirmed that metabolism of C-1311 directed to drug elimination was not dependent on hepatic cytochrome P450 activity.

Preliminary results on detoxification metabolism by UGT showed that C-1311 but not C-1330 underwent O-glucuronidation on the hydroxyl group at position 8 of the imidazoacridinone ring and the possibility of N-glucuronidation of 5-alkylaminoimidazocridinones should be excluded. Furthermore, the formation of O-glucuronides of C-1311 might be a reason for its lower toxicity compared with that of C-1330. We also demonstrated that C-1311 was a selective substrate of the UGT1 family but not of UGT2B7. Therefore, the detoxification pathway of C-1311 with UGT in patients might be limited by interindividual differences in the expression of UGT isoenzymes.

To summarize, we have shown that under in vitro conditions the antitumor agent C-1311 is not a substrate of human cytochrome P450 isoenzymes. Experiments with rat and human liver enzymes and with HRN mice strongly confirmed this conclusion. In contrast, imidazoacridinones C-1311 and C-1330 significantly inhibited CYP1A2 and CYP3A4 activity. On the other hand, this work demonstrated that the major metabolite, Nω-oxide, found with rat and human microsomes resulted from catalysis of FMOs. Glucuronidation of C-1311 occurring on the hydroxyl group seems to play a significant role in detoxification.

In light of previous results (Mazerska et al., 2001, 2003) demonstrating that C-1311 was efficiently metabolized by myeloperoxidase and that this compound was selectively effective toward leukemia cells, together with results presented here, we propose that metabolic activation of C-1311 occurs in myeloid cells with myeloperoxidase or with other peroxidases, but elimination of the drug requires the presence of microsomal FMOs and UGTs. P450 isoenzymes do not play a significant role in this metabolism, and, in fact, CYP1A2 and CYP3A4 are inhibited by C-1311.

Wrote or contributed to the writing of the manuscript: Potega, Henderson, and Mazerska.

Acknowledgments

We are grateful to Monika Legowska for her assistance with P450 inhibition experiments and to Kerry Wilson for language correction of this article.

Footnotes

This work was supported in part by the Ministry of Science and Higher Education (Poland) [Grant N401 159 32/3045]; the British-Polish Young Scientists Programme [WAR/342/84] of British Council; and Cancer Research UK [C4639/A5661] (to C.R.W.).